The term gonad refers to the mammalian reproduction system, and includes both the testis and the ovary. The function of these glands is dependent upon the interrelationship of a number of hormones and proteins, many of which are found, in varying amounts, in both sexes.
The ovary is the essential female reproductive organ in which eggs are produced. In vertebrates there are commonly two ovaries, suspended from the dorsal surface of the broad ligaments, one on each side of the uterus. Adult human ovaries are composed of a fibrous vascular stroma in which are imbedded the Graafian follicles, which contain the eggs. The eggs are discharged by the bursting of the Graafian follicles on the surface of the ovary and are then immediately received into the mouth of the oviduct. Thereafter, the eggs flow through the fallopian tube to the uterus, covered by a mucous membrane known as the endometrium, where the fertilized ovum develops.
The egg cells or ova are periodically matured in the ovaries at intervals of approximately four weeks. At the end of each four week period, one egg reaches maturity and passes into one of the fallopian tubes. The egg descends gradually and remains viable for a short while, and during this time fertilization may take place.
Within the ovary, there is a layer of cells called the germinal epithelium. Here, the potential egg begins its existence and continues to develop until a primary follicle, i.e., a group of cells isolated from the main layer, is formed around the potential egg. During a lifetime, each human ovary forms between 200,000 and 400,000 follicles. Of all these potential eggs only a few develop into mature eggs, most of them degenerating. The primary follicle that does not degenerate increases in size, and the egg cell itself enlarges up to thirty times its original size.
Other changes occur in the areas adjacent to the follicle. As the follicle matures, it moves toward the surface of the ovary and when the maturation process is complete, the follicle protrudes from the surface. At this time, ovulation occurs, i.e., the follicle bursts and the egg is expelled from the surface of the ovary.
The developing follicle produces sex hormones by metabolizing pre-hormones using a series of enzymes: 3.beta.-ol dehydrogenase, 17.alpha.-hydroxylase, hydroxysteroid dehydrogenase, and aromatase. Aromatase is a central enzyme in the production of sex hormones referred to as estrogens (estradiol, estrone and estriol).
Estrogenic hormones play a particularly important role in both the menstrual cycle and the reproductive cycle. Although 17.beta.-estradiol is the principal estrogenic hormone, a number of other estrogenic substances have been isolated, including estriol and estrone. These hormones induce the growth of the vaginal epithilium and secretion of mucous by the glands of the cervix and initiate the growth of the endometrium.
The corpus luteum, which fills a ruptured Graafian follicle in the mammalian ovary, produces at least three hormones, progesterone, 17.beta.-estradiol and relaxin. Progesterone acts to complete the proliferation of the endometrium, which was initiated by the estrogenic hormones, and to prepare it for the implantation of the ovum.
This reproductive cycle is well regulated as long as the production and secretion of both the sex hormones from the ovaries and the gonadotropic hormones of the pituitary gland are within normal limits. The anterior lobe of the pituitary, by manufacturing and secreting the gonadotropic hormones, controls the production of the sexual hormones in the ovaries and stimulates the development of the reproductive organs and the maintenance of their structure. The ovaries, under control of the gonadotropic hormones, produce the female sexual hormones. In turn, the rate of production of gonadotropins by the pituitary is influenced by the production of sex hormones. The effects are mutual and the two glands maintain an exact balance in hormone production.
More specifically, the follicle-stimulating hormone (FSH) from the pituitary stimulates the Graafian follicles, which thus produce estrogens. Estrogens not only inhibit FSH production through negative feedback on the pituitary, but also stimulate the pituitary to increase its production of luteinizing hormone (LH) through positive feedback. This hormone (LH) in turn brings about ovulation of the Graafian follicle. After the ova are discharged, the LH stimulates the empty follicle, now the corpus luteum, to produce progesterone. This hormone brings about the changes in the reproductive organs required for the development of the embryo. The progesterone then partly inhibits the pituitary from producing more LH. Thus, there is no further ovulation. The subsequent fall in progesterone then releases the pituitary inhibition allowing for the production of FSH to being the process anew.
When pregnancy occurs, the placenta of the embryo itself produces human chorionic gonadotropin (hCG), which stimulates the continuing production of progesterone from the corpus luteum, thus preventing menstruation and stimulating the continuing development of the uterus. This progesterone also inhibits further ovulation in spite of the presence of the hCG from the placenta.
The gonadotropic hormones have been determined to be proteins, with variable amounts of carbohydrates, and their structures are known. The molecular weight of human LH is about 26,000, and that of human FSH is about 30,000. The cellular response to gonadotropic hormones is translated through cellular receptors. These receptors are located within the cell membrane and are specific for each gonadotropin, thus, LH only activates LH receptors and FSH only activates FSH receptors.
Non-abortifacient means for the avoidance of pregnancy include oral contraceptive medications which contain estrogen and/or progesterone-like steroidal sex hormones. These medications, by raising the level of sex hormones in the blood stream, generate a cervical mucous which is hostile to spermatazoa. With increased levels of such hormones the endometrium tends to resist implantation of the fertilized ova. Further, the excess hormones provided by oral contraceptives directly inhibit the release of LH and FSH by the pituitary. As such, the ovarian cycle is disrupted and ovulation does not occur. The complications of such steroidal contraceptive medications, e.g., nausea, vomiting, weight gain, hypertension and tumor stimulation, adverse effects on calcium and phosphate metabolism, are well known and need not be discussed at length. However, these side effects result not only from the abnormally high levels of steroidal hormones in the blood stream, but from disruption of the hormonic homeostatis of the organism, i.e., the cycle of hormone adjustment between the ovary and the pituitary gland.
Accordingly, it has been a desideratum to provide a contraceptive medication which permits the regulation of the ovarian process without an accompanying disruption of the hormonic homeostatis of the organism.
While sex hormones are commonly referred to as male (androgenic) and female (estrogenic) hormones, these substances, including the proteinaceous substances associated therewith, are each important in the regulation of both the male and the female reproductive systems. For example, the luteinizing hormone (LH) and follicle stimulating hormone (FSH) play important roles in the regulation of the testis. These gonadotropins are synthesized and released in the male pituitary under the regulation of a hypothalamic peptide (luteinizing hormone releasing hormone) which is synthesized in the hypothalamus. LH binds to the surface receptors on the Leydig cells and promotes increased testosterone synthesis. It should be noted that testosterone synthesis is also regulated by the prevailing estradiol concentration, with high estradiol levels decreasing testosterone synthesis.
Spermatogenesis is a complex event where primitive germ cells (the spermatogonia) proceed through multiple cell divisions, in an orderly progression from spermatogonia to spermatocyte to spermatid, in order to form mature spermatozoa. In this progression, the intergenderal communality of the regulating hormones and proteins in the gonads is demonstrated by the fact that estrogen is a necessary component in the conversion of spermatogonia to spermatocytes. Further, the enzyme aromatase is a central enzyme in the conversion of androgens to estrogens. It should be noted that aromatase is a paracrine (intragonadal) enzyme that has been shown to be critical to the regulation of fertility in both the male and female gender of mammalian species.
It has also been a desideratum to provide a facile method for the regulation of fertility in both the male and female gender of mammalian species. It should be understood that the regulation of fertility, as employed herein, refers to a method and/or a composition of matter associated therewith, which enables either an increase or a decrease in mammalian fertility, that is, fertility control as opposed to mere contraception.
The present invention accomplishes the foregoing objectives by providing a protein moiety which enables the intragonadal modulation of the level of aromatase in a mammalian host and thus can regulate the maturation of ovarian follicles and the production of viable ova and regulate spermatogenesis and the production of mature spermatozoa without disturbing the normal level of sex hormones in the body, and permit the evaluation and diagnosis of gonadal function and dysfunction.
According to the invention a purified, nonsteroidal proteinaceous material or moiety having a measurable molecular weight of up to about 20,000, an isoelectric point of from about pH 3.5 to about pH 7.0 is provided, and is characterized by having the biological effect of inhibiting aromatase activity as defined by the extent of the conversion of androgens to estrogens. The protein inhibits intragonadal aromatase activity, modulates the intragonadal activity of 3.beta.-ol dehydrogenase, inhibits the development of granulosa cell LH receptors and prevents the maturation of mature ova and the production of mature spermatozoa. The term protein moiety, as used herein, refers to a protein, proteins or functional operative groups thereon which produce the described results. The production and activity of the protein moiety is interspecies, and is effective in both monotocous and polytocous mammals. Further, methods are provided for the isolation, purification and production of the protein, and for the use thereof in the regulation of fertility control.
In another aspect of the invention, antibodies are provided which inhibit the natural production of the protein with a corresponding increase in aromatase activity resulting in the promotion of follicular development, ovum maturation and spermatogenesis. Thus, mammalian fertility may be controlled substantially independently of exogenous sex hormones and without modulating extragonadal hormone levels. In yet another aspect of the invention, methods are provided for the use of antibodies to the protein for the quantification of the level of the protein in body fluids, thus permitting the evaluation and diagnosis of gonadal function and dysfunction.
For convenience, the intragonadal and follicular regulating protein is referred to herein as FRP.